Stabilization of thermolysin in aqueous solution

ABSTRACT

The present invention deals with the proteolytic enzyme thermolysin which tends to be unstable in aqueous solution. The invention provides methods and compositions to enhance the stability of dissolved thermolysin in aqueous solution. Thermolysin, crude thermolysin or a lyophilisate containing thermolysin and one or more salts, is contacted with an aqueous buffer with a low salt concentration and a first solution is formed. Subsequently, a further salt in solid form is added and dissociated, thereby forming a second solution comprising thermolysin in a stabilized form.

RELATED APPLICATIONS

This application claims priority to EP 08015100.4 filed Aug. 27, 2008.

FIELD OF THE INVENTION

The present invention pertains to the field of biochemistry. The presentinvention deals with the proteolytic enzyme thermolysin which tends tobe unstable in aqueous solution. The invention provides methods andcompositions to enhance the stability of dissolved thermolysin inaqueous solution. Thermolysin, crude thermolysin or a lyophilisatecontaining thermolysin and one or more salts, is contacted with anaqueous buffer with a low salt concentration and a first solution isformed. Subsequently, a further salt in solid form is added anddissociated, thereby forming a second solution comprising thermolysin ina stabilized form.

BACKGROUND OF THE INVENTION

Thermolysin [EC 3.4.24.27; CAS registry number 9073-78-3] is athermostable neutral metalloproteinase (also referred to herein as“neutral protease”) produced in the culture broth of Bacillusthermoproteolyticus (Endo, S., J., Ferment. Technol. 40 (1962) 346-353;Matsubara, H., Feder, J., in: 3rd ed., Boyer, P., D., (Ed.), TheEnzymes, vol. 3, Academic Press, New York, 1971, pp. 721-795). Itrequires one zinc ion for enzyme activity and four calcium ions forstructural stability (Latt, S., A., et al. Biochem. Biophys. Res.Commun. 37 (1969) 333-339; Feder, J., et al. Biochemistry 10 (1971)4552-4556; Tajima, M., et al. Eur. J. Biochem. 64 (1976) 243-247) andcatalyzes specifically the hydrolysis of peptide bonds containinghydrophobic amino acid residues (Morihara, K., Tsuzuki, H., Eur. J.Biochem. 15 (1970) 374-380; Inouye, K., et al. Biochem. J. 315 (1996)133-138). Thermolysin is widely used for the peptide bond formationthrough reverse reaction of hydrolysis (Oyama, K., et al., J. Chem. Soc.Perkin 11 (1981) 356-360; Nakanishi, K., et al., Ann. N.Y. Acad. Sci.613 (1990) 652-655; Trusek-Holownia, A., J. Biotechnol. 102 (2003)153-163). The npr gene that encodes thermolysin was isolated from B.thermoproteolyticus (O'Donohue, M., J., et al., Biochem. J. 300 (1994)599-603). Sequence analysis reveals that thermolysin is synthesized as apre-proprotein consisting a signal peptide (28 residues), a prosequence(204 residues), and a mature sequence (316 residues) (O'Donohue, M., J.,et al., supra). The prosequence acts as an intramolecular chaperoneleading to an autocatalytic cleavage of the peptide bond linking the proand mature sequences (O'Donohue, M., J., et al., J. Biol. Chem. 271(1996) 26477-26481; Marie-Claire, C., et al., J. Biol. Chem. 273 (1998)5697-5701; Marie-Claire, C., et al., J. Mol. Biol. 285 (1999)1911-1915).

The theoretical extinction at 280 nm of intact thermolysin in water canbe calculated using the “ProtParam tool” which is publicly available viathe internet. ProtParam is a tool which allows the computation ofvarious physical and chemical parameters for a given protein stored inSwiss-Prot or TrEMBL or for a user entered sequence. The computedparameters include the molecular weight, theoretical pI, amino acidcomposition, atomic composition, extinction coefficient, estimatedhalf-life, instability index, aliphatic index and grand average ofhydropathicity. Accordingly, the theoretical absorbance value in waterof A (1 mg/ml), at 280 nm of 1.696 can be calculated.

The manufacturer of thermolysin (Daiwa Kasei K.K., Japan) referring toOhta, Y et al. (J. Biol. Chem. 241 (1966) 5919-5925) indicates anabsorbance of A (1 mg/ml), of 1.765 at 280 nm in 50 mM TrisHCl buffer,pH 7.

Inouye, K., et al. (J. Biochem. 123 (1998) 847-852) discloses anabsorbance value A (1 mg/ml) of 1.83, determined at 277 nm and 25° C.for thermolysin Lot T8BA51 (Daiwa Kasei K.K., Osaka, Japan) in 10 mMCaCl₂, 40 mM TrisHCl, pH 7.5.

Thermolysin can be obtained as a lyophilisate from commercial suppliers.Daiwa Kasei K.K. (Japan) distributes a thermolysin with a molecularweight of 34,600 Da (Daltons), a pH optimum at pH 8.0, and a temperatureoptimum in the range of 65° C. and 70° C. According to the manufacturer,the enzyme is stable in a pH range of pH 5.0 and pH 8.5. A solubility of0.02% in dilute buffer solution is indicated. Twice crystallizedthermolysin can be purchased as a freeze-dried amorphous powder, whereinthe enzyme protein in the dried matter is 60% [w/w] or higher. The driedmatter additionally contains anhydrous calcium acetate (about 20% [w/w])and anhydrous sodium acetate (about 10% [w/w]). For furthercrystallization the manufacturer describes a method comprising the stepsof suspending the lyophilisate at a concentration in the range of 1%[w/v] and 5% [w/v] in an aqueous solution of 0.01 M. Calcium-acetate.The suspended material is dissolved by adding drop wise enough 0.2Nsodium hydroxide under agitation to bring the pH of the aqueous solutionto a value in the range of pH 11.0 and pH 11.4. After removal of anyundissolved residue, the pH of the solution is adjusted to pH 6.0 with0.2N acetic acid. The crystallization is usually complete inapproximately 2 days. The whole process is performed at a temperature inthe range of about 0° and 2° C.

Preparations of thermolysin are also available from Daiwa Kasei K.K.(Japan) under the trade name Thermoase.

EP 0 640 687 discloses an aqueous solution of 7 mM CaCl₂ and 1.75 M NaClin which Thermoase was dissolved to result in a concentration of about36 mg/ml. The purity of thermolysin in the dry Thermoase powder wasabout 20%. Taking purity into account, the concentration of thermolysinin the aqueous solution was about 7 mg/ml.

Inouye, K., et al. (J. Biochem. 123 (1998) 847-852) reported thatthermolysin is a sparingly soluble protein. It was suggested that thesurface of the protein is hydrophobic to a large extent and the factthat thermolysin can be purified efficiently by way of hydrophobicinteraction chromatography supports this notion (Inouye, K., et al.,Protein Expression and Purification 46 (2006) 248-255). As a consequenceof its low solubility, the enzyme has a strong tendency to precipitatewithin hours from freshly prepared solutions.

Inouye, K. et al. (J Biochem. supra) further demonstrated that thesolubility of thermolysin in an aqueous solvent can be increased ifcertain neutral salts are dissolved in the solvent when it is contactedwith a lyophilized preparation of thermolysin. The effect was shown tobe dependent on (1) temperature, (2) the particular neutral salt presentin the solvent, and (3) the concentration of the respective neutralsalt. In the document of Inouye, K. et al. (J. Biochem. supra) FIG. 2discloses the results of a series of experiments in which an excessiveamount of thermolysin lyophilized powder(three-times-crystallized-and-lyophilized preparation of thermolysin(Daiwa Kasei K.K., Osaka, Japan; Lot T8BA51; used without furtherpurification) was mixed with a “standard buffer” (10 mM CaCl2, 40 mMTrisHCl, pH 7.5) which additionally contained a salt at a predeterminedconcentration (in the range of 0.5 M and 5 M). The concentration ofdissolved protein was determined spectrophotometrically using anabsorbance value, A (1 mg/ml), at 277 nm of 1.83 and a molecular mass of34.6 kDa.

Tables 1-4 reproduce the approximate numerical values indicating theconcentrations of dissolved protein as graphically depicted in FIG. 2 ofInouye, K. et al. (J. Biochem. supra), at two different temperatures (0°C. and 37° C.). The protein concentrations tabulated are in mg/ml. Ineach table the salts dissolved in the standard buffer are indicated aswell as their respective concentrations.

TABLE 1 Concentrations of protein (in [mg/ml]) soluble at 0° C. instandard buffer containing salt concentration of salt in standard buffer0.5 M 1.0 M 1.5 M 2.0 M 2.5 M salt NaCl ~6.4 ~8.9 ~10.3 ~12.2 ~11.6 KCl~4.5 ~6.3 ~7.5 ~6.5 ~5.3 LiCl ~1.9 ~3.3 ~4.5 ~5.3 ~6.6 NaBr ~4.4 ~6.6~15.6 ~25.3 ~38.4 NaJ ~5.5 ~7.8 ~20.3 ~29.2 ~32.5

TABLE 2 Concentrations of protein (in [mg/ml]) soluble at 0° C. instandard buffer containing salt concentration of salt in standard buffer3.0 M 3.5 M 4.0 M 4.5 M 5.0 M salt NaCl ~9.5 ~8.1 ~6.9 ~5.3 ~3.8 KCl~5.2 ~4.4 ~4.1 ~3.4 ~2.5 LiCl ~8.0 ~11.1 ~14.7 ~21.6 ~26.3 NaBr ~40~36.6 ~30 ~27.8 ~25.3 NaJ ~34.4 ~37.5 ~38.6 ~42.3 § § out of detectionrange

TABLE 3 Concentrations of protein (in [mg/ml]) soluble at 37° C. instandard buffer containing salt concentration of salt in standard buffer0.5 M 1.0 M 1.5 M 2.0 M 2.5 M salt NaCl ~2.7 ~3.8 ~5.5 ~7.5 ~8.8 KCl~1.6 ~3.1 ~3.2 ~4.2 ~3.4 LiCl ~0.9 ~2.2 ~2.5 ~3.1 ~4.5 NaBr ~3.4 ~5.0~9.7 ~13.3 ~18 NaJ ~3.4 ~6.2 ~18 ~24.5 ~34.4

TABLE 4 Concentrations of protein (in [mg/ml]) soluble at 37° C. instandard buffer containing salt concentration of salt in standard buffer3.0 M 3.5 M 4.0 M 4.5 M 5.0 M salt NaCl ~7.4 ~5.5 ~3.1 ~2.3 ~1.0 KCl~3.1 ~2.8 ~2.2 ~1.9 ~0.6 LiCl ~7.0 ~8.8 ~11.3 ~18 ~22.3 NaBr ~22.2 ~24.4~26.3 ~29 ~33.1 NaJ ~36.7 ~38.1 ~35.3 ~38.6 ~38.0

Accordingly, for selected salts the highest concentrations of solubleprotein were each about

 8.8 mg/ml at 37° C. in the presence of 2.5 M   NaCl, 12.2 mg/ml at 0°C. in the presence of 2 M NaCl,  4.2 mg/ml at 37° C. in the presence of2 M KCl,  7.5 mg/ml at 0° C. in the presence of 1.5 M   KCl, 22.3 mg/mlat 37° C. in the presence of 5 M LiCl, 26.3 mg/ml at 0° C. in thepresence of 5 M LiCl, 33.1 mg/ml at 37° C. in the presence of 5 M NaBr,  40 mg/ml at 0° C. in the presence of 3 M NaBr, 38.6 mg/ml at 37° C. inthe presence of 4.5 M   NaJ, and  >45 mg/ml at 0° C. in the presence of5 M NaJ.

Thermolysin is an aggressive protease which in solution undergoesautoproteolytic attack. Thus, both crystallized and freeze-driedpreparations of thermolysin as well as solutions of such preparationscontain amounts of different autoproteolytic fragments of thermolysin.

In order to limit autoproteolytic attack, low temperatures are appliedto solutions containing thermolysin. However, enzymatic activity is onlyreduced (i.e. some proteolytic activity is still present) under suchconditions, and not brought to a complete halt. In this regard it isnoted that Inouye, K. et al, (J. Biochem. supra) determines proteincontent of solutions without any purification step. The proteinconcentrations detected therefore correspond to mixtures of intactthermolysin and degradation fragments thereof.

In view of the state of the art it is an object of the present inventionto provide methods and compositions with a stabilized form ofthermolysin in an aqueous solution. By providing a stabilized form, thetendency of thermolysin to precipitate is reduced, and solutions of theenzyme remain in a homogeneous state for a prolonged time.

The inventors have unexpectedly found that by dissolving thermolysinfirst in a buffer with a low ion concentration and then adding a saltand dissolving the salt in the solution which already containsthermolysin surprisingly allows to form a solution with a highconcentration of thermolysin. At the same time, under such conditionsaccording to the invention dissociated thermolysin is stabilized in thesolution, i.e. the solution remains clear for an increased amount oftime during which no precipitate is formed.

The invention provides significant benefit when amounts of thermolysinhave to be kept in solution for dispensing aliquots thereof, or formaking blends with preparations of other enzymes, such as collagenaseenzymes. Such blends of proteolytic enzymes are of particular use in thedissociation of organ tissue for the separation of subsets of cells fromthe tissue.

SUMMARY OF THE INVENTION

A first aspect of the invention is a method for preparing a solution ofthermolysin (EC 3.4.24.27) in which the dissolved thermolysin is in astabilized form, the method comprising the first step (P) of mixing asolid preparation comprising thermolysin with an aqueous solvent andmaking a first solution, wherein the first solution comprises (i) abuffer salt capable of maintaining a pH in the range of pH 4.5 and pH 9,(ii) one or more salts, and (iii) thermolysin, and in the first solutionthe aggregate concentration of the one or more salts including thebuffer salt or salts is in the range of about 0.1 mM and about 150 mM,wherein the method further comprises the subsequent step (Q) of addingto the first solution a measured amount of a further salt, whereby thesalt is selected from the group consisting of NaCl, NaBr, NaNO₃, NaJ,KCl, LiCl, MgCl₂, CaCl₂, and a mixture thereof, and dissolving thefurther salt, thereby preparing a second solution in which the dissolvedthermolysin is in a stabilized form.

Another aspect of the invention is a liquid composition comprisingwater, thermolysin in a dissociated form, a dissociated buffer saltcapable of maintaining a pH in the range of pH 4.5 and pH 9, and adissociated salt selected from the group consisting of NaCl, NaBr,NaNO₃, NaJ, KCl, LiCl, MgCl₂, CaCl₂, and a mixture thereof, wherein thecomposition is a homogeneous solution for five hours or more, and thecomposition contains thermolysin at a concentration in the range ofabout 1 mg/ml and about 10 mg/ml.

A further aspect of the invention is a liquid composition comprisingwater, thermolysin in a dissociated form, a dissociated buffer saltcapable of maintaining a pH in the range of pH 4.5 and pH 9, and adissociated salt selected from the group consisting of NaCl, NaBr,NaNO₃, NaJ, LiCl, MgCl₂, CaCl₂, and a mixture thereof, wherein thecomposition is a homogeneous solution for five hours or more, and thecomposition contains thermolysin at a concentration in the range ofabout 10 mg/ml and about 23 mg/ml.

Yet, another aspect of the invention is the use of a compositionaccording to the invention for storage, transport, or dispensing ofthermolysin in dissociated form.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Results of a wavelength scan as described in Example 8. Theordinate indicates light intensity in arbitrary units (a.u.) measured bythe detector at 800 nm. The abscissa indicates the wavelength of theincoming light. (A) liquid sample containing the aqueous buffer; (B)liquid sample containing the colloid of thermolysin in the aqueousbuffer.

FIG. 2 Exemplary result of a chromatographic analysis of a solutioncontaining Thermoase lyophilisate obtained from the first step describedin Example 2, i.e. without/prior to a further diafiltration step.Thermoase lyophilisate was dissolved in an aqueous buffer containing 2.3M NaCl, 5 mM CaCl₂, 20 mM HEPES, pH 7.5; the protein content of thesolution was 8.2 mg/ml as determined photometrically at 280 nm. TheFigure shows a HPLC chromatogram a 50 μl sample of the homogeneoussolution. Five peak areas denoted (i) to (v) are marked. Conditions andparameters of HPLC are described in Example 2. Ordinate: mA.U.;abscissa: retention time in [min].

FIG. 3 Exemplary result of a chromatographic analysis of a solutioncontaining Thermoase lyophilisate obtained from the second stepdescribed in Example 2, i.e. including/after the diafiltration step.Following diafiltration, thermoase was in an aqueous buffer containing170 mM NaCl, 5 mM CaCl₂, 20 mM HEPES, 7.5; the protein content of thesolution after diafiltration was 4.9 mg/ml as determined photometricallyat 280 nm. The Figure shows a HPLC chromatogram a 50 μl sample of thehomogeneous solution. Three peak areas denoted (vi) to (viii) aremarked. Conditions and parameters of HPLC are described in Example 2.Ordinate: mA.U.; abscissa: retention time in [min].

DETAILED DESCRIPTION OF THE INVENTION

Certain terms are used with particular meaning, or are defined for thefirst time, in this description of the present invention. For thepurposes of the present invention, the terms used are defined by theirart-accepted definitions, when such exist, except that when thosedefinitions conflict or partially conflict with the definitions setforth below. In the event of a conflict in definition, the meaning of aterm is first defined by any of the definitions set forth below.

The term “comprising” is used in the description of the invention and inthe claims to mean “including, but not necessarily limited to”.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “a compound” means one compound or more thanone compound.

When designating a range of numerical values such as, but not limitedto, a concentration range, the range is indicated by a first value n1and a second value n2 (e.g. “a range of n1 and n2”). The lower boundaryof the designated range is understood as being the value equal to orhigher than the first value. The higher boundary of the designated rangeis understood as being the value equal to or lower than the secondvalue. Thus, a value x in the designated range is given by n1≦x≦n2. Whena range is indicated using the word “between”, the upper and the lowerboundary are understood as being included in the interval. Thus; theexpression “a value x between n1 and n2” is understood as n1≦x≦n2.

If not stated otherwise, it is understood that the term “about” and thecharacter “˜” in combination with a numerical value n (“about n”, “˜n”)indicates a value x in the interval given by the numerical value±5% ofthe value, i.e. n−0.05*n≦x≦n+0.05*n. In case the term “about” or thecharacter “˜” in combination with a numerical value n describes apreferred embodiment of the invention, the value of n is most preferred,if not indicated otherwise.

A “mixture” is a substance made by combining two or more differentmaterials with no chemical reaction occurring. The objects do not bondtogether in a mixture. A mixture can usually be separated back into itsoriginal components. Mixtures are the product of a mechanical blendingor mixing of chemical substances like elements and compounds, withoutchemical bonding or other chemical change, so that each ingredientsubstance retains its own chemical properties and makeup. While thereare no chemical changes in a mixture, physical properties of a mixture,such as its melting point, may differ from those of its components.Mixtures are either homogeneous or heterogeneous.

Homogeneous mixtures are mixtures that have definite, consistentproperties. Particles are uniformly spread. For example, any amount of agiven mixture has the same composition and properties. A homogeneousmixture is a uniform mixture consisting of only one phase. For thepurpose of the invention, solutions of one or more dissociated salts arenon-limiting examples for homogeneous mixtures.

A solution is a homogeneous mixture of one or more substances (thesolutes) dissolved (i.e. dissociated) in another substance (thesolvent). A common example would be a solid dissolving into a liquid(i.e. salt or protein dissolving in water). Solubility is a compoundproperty. Depending on the conditions, the amount of a substance thatcan dissolve in a solvent or a solution can be variable.

Non-limiting examples for non-homogeneous (heterogeneous) mixtures are acolloid and a suspension. In the context of the invention, a suspensionis understood as being a heterogenous fluid containing solid particlesthat are sufficiently large for sedimentation. Unlike colloids, thesuspended particles settle over time if left undisturbed. Thisdistinguishes a suspension from a colloid in which the suspendedparticles are smaller and do not settle.

In a solution, the dissolved substance does not exist as a solid, andsolute(s) and solvent are homogeneously mixed. The term “stability” of asolution refers to the tendency of the dissolved substance to remain inthe dissolved state. That is to say, the term refers to the ability ofthe solution to remain homogeneous during a given time interval.Stability can therefore be characterized in a quantifying way bydetermining said time interval. Thus, the dissolved substance in a firstsolution characterized by a lower stability exhibits a higher tendencyto precipitate or form a colloid or a suspension, as opposed to a secondsolution characterized by a higher stability in which said tendency islower. As a consequence, after a certain amount of time said firstsolution becomes a heterogeneous mixture whereas said second solutionremains a homogeneous mixture.

Under certain conditions the stability of a solution can be increased,that is to say the tendency of a dissolved substance to precipitate isreduced. For the purpose of the present invention, a substance with areduced tendency to precipitate is referred to as being in “a stabilizedform”.

Turbidity is a measure of water cloudiness caused by the presence ofparticles in a suspension or a colloid. There are several practical waysof determining turbidity, the most direct being some measure ofattenuation (that is, reduction in strength) of light as it passesthrough a sample column of water. Thus, one way to determine turbidityis visual inspection, i.e. inspection by eye.

Another way of determination is measurement of light attenuation with aphotometer. In this regard, the term “Optical density” (also referred toas “OD”) denotes a unitless measure of the transmittance of an opticalelement for a given length at a given wavelength λ:OD₈₀=log₁₀ O=−log₁₀ T=−log₁₀(I/I ₀)whereby

-   -   O=the per-unit opacity    -   T=the per-unit transmittance    -   I₀=the intensity of the incident light beam.    -   I=the intensity of the transmitted light beam.

The higher the optical density, the lower the transmittance. Owing tothe scattering of a light beam focused on the particles the opticaldensity of a suspension or a colloid is increased compared to a clearsolution.

A preferred way to determine turbidity is to measure the scatteredlight. To this end, a light scattering photometer is used frequently.Depending on the direction from which light scatter is detected andquantified, there are several types of scattered light photometers knownto the art. In principle, all can be used for quantitative assessment ofturbidity in liquid samples. The term “light scatter” collectivelyincludes both the scatter of light waves by particles in the sample, aswell as reflection by particulate matter in the sample. Back Scatter isdefined as less than 90°, toward the light source. Forward scatter isdefined as less than 90° away from, or in the same general direction asthe light source. A majority of turbidity units of measure used todayare based on 90° side scatter measurement techniques.

The intensity of the scattered light depends on the amount of thenon-dissolved (particulate) matter in the heterogeneous mixture and canbe described by Formula 1:F=I ₀·Φ·(2.303·ε·c·d)  (Formula 1)whereby

-   -   F is the intensity of the scattered light    -   I₀ is the intensity of the incoming light beam    -   Φ is the ratio of emitted versus absorbed photons    -   ε is the molar absorption coefficient of the particulate        substance in the mixture    -   c is the amount of the particulate substance per volume of the        liquid sample (heterogeneous mixture) in the cuvette    -   d is the thickness of the space in the cuvette

For the purpose of the invention, 90° side scatter measurements are madewith a fluorescence photometer to determine cloudiness of heterogeneousmixtures containing thermolysin as particulate matter. Typically, suchmixtures are colloids.

“Crude thermolysin” in the sense of the invention is a protein mixtureconsisting mainly of substantially undegraded (=intact) thermolysin andadditionally of degradation products, typically resulting fromautoproteolytic attack. Usually, about 70% of crude thermolysin is foundsubstantially undegraded, while about 24% of crude thermolysin consistsof different degradation products which retain proteolytic activity (todifferent degrees), and about 6% are proteolytically inactive fragmentsand further impurities. Thermoase is a preparation of crude thermolysinwhich is used to exemplify the advantageous effects of the presentinvention. However, the present invention is not limited to the use ofThermoase preparations, and other preparations of the enzyme can be usedto practice the invention.

Thermoase is a lyophilisate with a protein content which is in the rangeof about 30% [w/w] and about 35% [w/w]. The protein in the lyophilizateconsists of “crude thermolysin”.

From the state of the art it is known that solubility of thermolysinincreases if a salt, preferably a dissociated neutral salt is present inthe aqueous buffer to be used as solvent of dry thermolysin, or of a drycomposition containing thermolysin. However, aqueous solutions ofthermolysin are unstable in that the dissolved thermolysin undergoes atransition which reduces its solubility. The precise nature of thetransition is unclear but one can reasonably speculate that amino acidresidues of one or more hydrophobic domain(s) of thermolysin play a rolein this process. Due to the transition, solubility of thermolysindecreases. As a result, a freshly prepared clear solution of the enzymebecomes opaque and a substantial portion of the protein eventuallyprecipitates. An example for the lack of stability is given in Example6, Tables 13 and 14, No. 1-3. Even at reduced concentrations of thelyophilisate and in the presence of about 1.1 M NaCl, thermolysin has astrong tendency to precipitate after being brought in solution.

The inventors have surprisingly found that the transition can besuppressed and thermolysin can be stabilized in solution. To this end,thermolysin, crude thermolysin or a lyophilisate containing thermolysinand one or more salts is contacted with an aqueous buffer with a lowsalt concentration to form a solution which is stable only for a shorttime interval. Subsequently, a further salt in solid form is added anddissociated in the solution afterwards. After this step thermolysin insolution is in a stabilized form which is characterized by asignificantly reduced tendency to precipitate.

According to the invention, before the aqueous buffer with the low saltconcentration is contacted with thermolysin, crude thermolysin or alyophilisate containing thermolysin, the concentration of dissociatedsalts, including buffer salt, in the buffer is preferably lower than 150mM, preferably in the range of 0.1 mM and 150 mM. Depending on thepreparation of thermolysin, the salt concentration of the aqueous bufferincreases to the extent of salt being present in the thermolysincontaining solid preparation dissociated in the buffer.

The salt in solid form preferably is a neutral salt with the exceptionof an inorganic sulfate salt. A preferred solid salt is selected fromthe group consisting of NaCl, NaBr, NaNO₃, NaJ, KCl, LiCl, MgCl₂, CaCl₂,and a mixture thereof.

The first main step is the formation of a clear solution containingthermolysin. In case the preparation of thermolysin does not allow toform a homogeneously clear solution, an additional clearing step isnecessary. For example, the solution can be cleared by way offiltration, centrifugation or equivalent means.

As the second main step, the salt in solid form has to be added to thefreshly prepared clear solution of thermolysin. At a temperature in therange of about 2° C. and about 8° C. the salt in solid form is addedpreferably not more than 30 min after the clear solution of thermolysinis obtained. Shorter periods such as not more than 15 min, 10 min, and 5min are more preferred.

Using in the thermolysin solution a total concentration in the range ofabout 1.5 M and 3.5 M of the dissociated salt, thermolysin in solutionbecomes stabilized and the solution remains a clear homogeneous mixturefor up to 16 h or even longer.

Surprisingly, this solution can be even diafiltrated against a bufferwith a lower salt concentration; in such a process the concentration ofthe dissociated salt which was added previously can be lowered (seeExample 2). Diafiltration in this regard is a crossflow filtrationprocess allowing for the transfer of low molecular weight species, waterand/or solvents through a membrane without changing the solution volume.This process is used for purifying retained large molecular weightspecies (i.e. substantially intact thermolysin), while low molecularweight species including proteolytic fragments of thermolysin areremoved. The procedure of diafiltration at the same time allows forbuffer exchange, thereby simply changing the properties of a givensolution prior to the diafiltration process.

Even under these conditions (i.e. during a diafiltration process)thermolysin remains in a stabilized form, that is to say thermolysinremains stably in solution. The same effect is observed for frozenthermolysin solutions according to the invention after thawing.

Therefore, the invention particularly provides the means; compositionsand conditions to manipulate homogeneous solutions of thermolysin for anextended amount of time under reproducible conditions. This isparticularly useful when thermolysin is blended with other enzymes orwhen thermolysin solutions are dispensed as aliquots, e.g. usingautomated devices.

Yet, in more detail, the present invention comprises the followingitems:

-   1. A method for preparing a solution of thermolysin (EC 3.4.24.27)    in which the dissolved thermolysin is in a stabilized form, the    method comprising the first step (P) of mixing a solid preparation    comprising thermolysin with an aqueous solvent and making a first    solution, wherein the first solution comprises    -   (i) a buffer salt capable of maintaining a pH in the range of pH        4.5 and pH 9,    -   (ii) one or more salts, and    -   (iii) thermolysin,    -   and in the first solution the aggregate concentration of the one        or more salts including the buffer salt or buffer salts is in        the range of about 0.1 mM and 500 mM, and    -   wherein the method further comprises the subsequent step (Q) of        adding to the first solution a measured amount of a further        salt, whereby the salt is selected from the group consisting of        NaCl, NaBr, NaNO₃, NaJ, KCl, LiCl, MgCl₂, CaCl₂, and a mixture        thereof, and dissolving the further salt,    -   thereby preparing a second solution in which the dissolved        thermolysin is in a stabilized form.-   2. The method according to item 1, wherein the first solution    obtained in step (P) is a homogeneous solution.-   3. The method according to any of the items 1 and 2, wherein in    step (P) the aqueous solvent comprises water and a buffer salt.-   4. The method according to any of the items 1 and 2, wherein in    step (P) the solid preparation comprising thermolysin additionally    comprises one or more salts.-   5. The method according to item 4, wherein in step (P) the aqueous    solvent is water.-   6. The method according to any of the items 1 to 5, wherein in    step (P) the solid preparation comprises thermolysin at a    concentration of about 20% [w/w] or higher.-   7. The method according to any of the items 1 to 5, wherein in    step (P) the solid preparation comprises thermolysin at a    concentration in the range of about 20% [w/w] and 100% [w/w], more    preferred in the range of about 20% [w/w] and about 80% [w/w], and    even more preferred in the range of about 20% [w/w] and about 60%    [w/w].-   8. The method according to item 6 or item 7, wherein in step (P) the    solid preparation comprises thermolysin at a concentration in the    range of about 20% [w/w] and about 50% [w/w], more preferred in the    range of about 20% [w/w] and about 40% [w/w], and even more    preferred in the range of about 20% [w/w] and about 30% [w/w].-   9. The method according to item 8, wherein in step (P) the solid    preparation comprises thermolysin at a concentration of about 20%    [w/w].-   10. The method according to any of the items 1 to 9, wherein in    step (P) the concentration of the preparation comprising thermolysin    in the aqueous solvent is in the range of about 1 mg/ml and about    100 mg/ml.-   11. The method according to item 10, wherein in step (P) the    concentration of the solid preparation is in the range of about 20    mg/ml and about 60 mg/ml, and more preferred in the range of about    25 mg/ml and about 50 mg/ml, and even more preferred about 30 mg/ml.-   12. The method according to any of the items 1 to 11, wherein in    step (P) the one or more salts (ii) in the first solution comprise a    salt selected from the group consisting of NaCl, Na₂SO₄, and a    combination thereof.-   13. The method according to item 4, wherein in step (F) the one or    more salts in the solid preparation comprising thermolysin are    selected from the group consisting of NaCl, Na₂SO₄, and a    combination thereof.-   14. The method according to item 13, wherein in step (P) the solid    preparation contains NaCl in the range of about 50% [w/w] and about    70% [w/w], and/or Na₂SO₄ in the range of about 0.5% [w/w] and about    7.5% [w/w], and more preferred the solid preparation contains NaCl    in the range of about 60% [w/w] and about 65% [w/w], and/or Na₂SO₄    in the range of about 3% [w/w] and about 6% [w/w].-   15. The method according to any of the items 12 to 14, wherein in    step (P) the solid preparation contains crude thermolysin.-   16. The method according to any of the items 1 to 15, wherein in    step (P) the concentration of sulfate ions in the first solution is    in the range of about 1 mM and about 10 mM, more preferred the    concentration of sulfate ions in the first solution is about 5 mM.-   17. The method according to any of the items 1 to 16, wherein in the    subsequent step (Q) the further salt is added in solid form.-   18. The method according to any of the items 1 to 17, wherein the    total concentration of the further salt in the second solution    obtained after step (Q) is in the range of about 1.5 M and about 5    M.-   19. The method according to item 18, wherein the total concentration    of the further salt in the second solution obtained after step (Q)    is in the range of about 2 M and about 3.5 M.-   20. The method according to item 19, wherein the total concentration    of the further salt in the second solution obtained after step (Q)    is in the range of about 2 M and about 2.5 M.-   21. The method according to item 19, wherein the total concentration    of the further salt in the second solution obtained after step (Q)    is about 2.3 M.-   22. The method according to any of the items 17 to 21, wherein the    concentration of sulfate ions in the second solution obtained after    step (Q) is lower than about 10 mM.-   23. The method according to item 22, wherein the concentration of    sulfate ions in the second solution obtained after step (Q) is in    the range of about 1 mM and about 10 mM, more preferred in the range    of about 1 mM and about 5 mM.-   24. The method according to any of the items 1 to 23, wherein in the    first solution obtained in step (P) the aggregate concentration of    the one or more salts including the buffer salt or salts is in the    range of about 100 mM and 500 mM, more preferred in the range of    about 300 mM and 400 mM.-   25. The method according to any of the items 1 to 24, wherein in    step (P) the aqueous solvent comprises Ca²⁺ ions.-   26. The method according to item 25, wherein in the aqueous solvent    the concentration of Ca²⁺ ions is in the range of about 0.1 mM and    about 10 mM, even more preferred in the range of about 1 mM and    about 10 mM.-   27. The method according to item 26, wherein in the aqueous solvent    the concentration of Ca²⁺ ions is about 5 mM.-   28. The method according to any of the items 1 to 27, wherein in    step (P) the aqueous solvent comprises a buffer salt at a    concentration in the range of about 0.1 mM and about 100 mM, and    more preferred in the range of about 1 mM and about 100 mM.-   29. The method according to item 28, wherein in the aqueous solvent    the concentration of the buffer salt is in the range of about 5 mM    and about 50 mM.-   30. The method according to item 29, wherein in the aqueous solvent    the concentration of the buffer salt is in the range of about 15 mM    and about 25 mM.-   31. The method according to item 30, wherein in the aqueous solvent    the concentration of the buffer salt is about 20 mM.-   32. The method according to any of the items 28 to 31, wherein the    buffer salt is selected from the group consisting of BES    (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Tris    (tris(hydroxymethyl)aminomethane),    BisTris(Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane),    BisTris propane (1,3-bis(tris(hydroxymethyl)methylamino)propane),    HEPES (N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid), MES    (2-(N-morpholino)ethanesulfonic acid), MOPS    (3-(N-morpholine)propanesulfonic acid), MOPSO    (3-morpholino-2-hydroxypropanesulfonic acid), PIPES    (Piperazine-1,4-bis(2-ethanesulfonic acid)),    TAPS(N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), TES    (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), TEA    (Triethanolamine), and Tricine    (N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine).-   33. The method according to any of the items 1 to 32, wherein in    step (P) the pH of the first solution is in the range of about pH    5.5 and about pH 8.5.-   34. The method according to item 33, wherein the pH is about pH 7.5.-   35. The method according to any of the items 1 to 34, wherein in the    second solution obtained after step (Q) the concentration of    substantially intact thermolysin is about 5 mg/ml or higher, and    wherein the thermolysin in solution is in a stabilized form.-   36. The method according to item 35, wherein in the second solution    obtained after step (Q) the concentration of substantially intact    thermolysin is about 5 mg/ml or higher, and lower than about 35    mg/ml.-   37. The method according to item 35, wherein the further salt is    selected from the group consisting of KCl, LiCl, NaCl, NaBr, NaJ,    NaNO₃, MgCl₂, CaCl₂, and a mixture thereof.-   38. The method according to item 37, wherein the concentration of    thermolysin in a stabilized form is about 10 mg/ml or higher, and    lower than about 35 mg/ml.-   39. The method according to item 38, wherein the concentration of    thermolysin in a stabilized form is about 20 mg/ml.-   40. A liquid composition comprising water, thermolysin in a    dissociated form, a dissociated buffer salt capable of maintaining a    pH in the range of pH 4.5 and pH 9, and a dissociated salt selected    from the group consisting of NaCl, NaBr, NaNO₃, NaJ, KCl, LiCl,    MgCl₂, CaCl₂, and a mixture thereof, wherein the composition    contains thermolysin at a concentration in the range of about 1    mg/ml and about 10 mg/ml.-   41. A liquid composition comprising water, thermolysin in a    dissociated form, a dissociated buffer salt capable of maintaining a    pH in the range of pH 4.5 and pH 9, and a dissociated salt selected    from the group consisting of NaCl, NaBr, NaNO₃, NaJ, LiCl, MgCl₂,    CaCl₂, and a mixture thereof, wherein the composition is a    homogeneous solution for five hours or more, and the composition    contains thermolysin at a concentration in the range of about 10    mg/ml and about 23 mg/ml.-   42. The liquid composition according either to item 40 or according    to item 41, the liquid composition being obtainable by a method    according to any of the items 1 to 39.-   43. The liquid composition according to any of the items 40 to 42,    wherein the dissociated thermolysin is in a stabilized form.-   44. The liquid composition according to any of the items 40 to 43,    wherein at a temperature in the range of about 2° C. and about 8° C.    and at least five hours after formation of the composition the    turbidity of the composition about equals the turbidity of a    reference solution, whereby said reference solution is composed of    the same dissociated ingredients at the same respective    concentrations as in the composition according to any of the items    41 and 42, and whereby the reference solution lacks thermolysin or    fragments thereof.-   45. The liquid composition according to any of the items 40 to 44,    obtained by a method according to any of the items 1 to 39 and    incubated for at a temperature in the range of about 2° C. and about    8° C., said composition being a homogeneous solution in an    incubation interval of 0-5 hours, even more preferred in an    incubation interval of more than 5 hours.-   46. The liquid composition according to any of the items 44 and 45,    wherein the dissociated thermolysin is substantially intact.-   47. The liquid composition according to any of the items 44 to 46,    wherein at a temperature of in the range of about 2° C. and about    8° C. for at least five hours after formation of the composition the    turbidity of the composition about equals the turbidity of a    reference solution, whereby said reference solution is composed of    the same dissociated ingredients at the same respective    concentrations as in the composition according to any of the items    44 to 46, and whereby the reference solution lacks thermolysin or    fragments thereof.-   48. A liquid composition comprising water, thermolysin in a    dissociated form, a dissociated buffer salt capable of maintaining a    pH in the range of pH 4.5 and pH 9, and dissociated NaCl at a    concentration below 500 mM, and wherein the composition contains    thermolysin at a concentration in the range of about 1 mg/ml and    about 10 mg/ml.-   49. The liquid composition according to item 48, wherein the pH of    the composition is in the range of pH 7 and pH 8, even more    preferred at about pH 7.5.-   50. The liquid composition according to any of the items 48 and 49,    wherein the composition comprises Ca²⁺ ions, more preferred Ca²⁺    ions at a concentration in the range of about 0.1 mM and about 10    mM, and even more preferred in the range of about 1 mM and about 10    mM.-   51. The liquid composition according to any of the items 48 to 50,    wherein in the composition the aggregate concentration of the    dissociated salts including the buffer salt or salts is in the range    of about 400 mM and about 200 mM, and more preferred the aggregate    concentration of the dissociated salts including the buffer salt or    salts is about 200 mM.-   52. The liquid composition according to any of the items 48 to 51,    wherein in the composition the concentration of dissociated sodium    ions is in the range of 100 mM and 250 mM, more preferred the    concentration of dissociated sodium ions is in the range of 150 mM    and 200 mM, and even more preferred the concentration of dissociated    sodium ions is about 170 mM.-   53. The liquid composition according to any of the items 48 to 52,    wherein the conductivity of the composition is about 20 mS/cm.-   54. The liquid composition according to any of the items 48 to 53,    wherein in the composition the concentration of substantially intact    thermolysin is in the range of 0.1 mg/ml and 10 mg/ml, more    preferred in the range of 1 mg/ml and 7.5 mg/ml, and even more    preferred between 1 mg/ml and 5 mg/ml, even more preferred 2.5 mg/ml    or 5 mg/ml.-   55. The liquid composition according to any of the items 48 to 54,    the liquid composition being obtainable by the method according to    any of the items 1 to 39, followed by a subsequent step of    diafiltrating the second solution obtained after step (Q) against a    diafiltration buffer containing a dissociated buffer salt capable of    maintaining a pH in the range of pH 4.5 and pH 9, and dissociated    NaCl at a concentration below 500 mM.-   56. The liquid composition according to item 55, wherein the    diafiltration buffer comprises Ca²⁺ ions at a concentration in the    range of about 0.1 mM and about 10 mM.-   57. The liquid composition according to any of the items 55 and 56,    wherein in the diafiltration buffer the aggregate concentration of    dissociated salts including the buffer salt or salts is in the range    of about 400 mM and about 200 mM, and more preferred the aggregate    concentration of the dissociated salts including the buffer salt or    salts is about 200 mM.-   58. Use of a liquid composition according to any of the items 40 to    57 for storage, transport, or dispensing of thermolysin, whereby the    thermolysin is in dissociated form.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Example 1 Preparation of Mixtures of an Aqueous Buffer and Thermolysinfrom a Thermoase Preparation

According to information provided by the manufacturer, about 60-65%[w/w] of the lyophilisate was NaCl. In addition, the lyophilizatecontained about 5% [w/w] Na₂SO₄ (decahydrate). An amount in the range ofabout 30% [w/w] and about 35% [w/w] of the Thermoase lyophilisate usedhere as well in the Examples further below consisted of crudethermolysin (see also Example 2). All of the working steps describedbelow were performed at a temperature of in the range of 2° C. and 8°C., if not indicated otherwise.

A volume of 8 l of the aqueous buffer (A) containing 2 M NaCl, 5 mMCaCl₂, 20 mM HEPES, pH 7.5 was prepared. An amount of 200 g dryThermoase lyophilisate was mixed with the aqueous buffer and the mixturewas stirred continuously. However, no homogeneous mixture was obtained,the mixture remained turbid and never became entirely clear. About 60min following the addition of the lyophilisate, the mixture becameincreasingly opaque and thermolysin started to precipitate.

Surprisingly, formation of an inhomogeneous mixture as obtained withbuffer (A) could be avoided by first dissolving the Thermoasepreparation in a buffer with low ionic strength and adding salt onlythereafter. Thus, in a first step a volume of 6.5 l of the aqueousbuffer (B) containing 5 mM CaCl₂, 20 mM HEPES, pH 7.5 was prepared. Anamount of 200 g dry Thermoase lyophilisate was dissolved in buffer (B)and a clear solution was obtained. Subsequently, as a second step, 935 gsolid NaCl was added and dissolved in the solution. As a third step, thevolume of the solution was adjusted to 8 l by adding a further volume ofaqueous buffer (B) and mixing by stirring. A homogeneous solution wasobtained.

Taking into account that about 60-65% [w/w] of the lyophilisate consistsof NaCl, the final NaCl concentration in the solution was about 2.3 M.The final concentration of crude thermolysin in solution was in therange of about 7.5 mg/ml and 8.8 mg/ml corresponding to a concentrationof substantially undegraded thermolysin of about 5.7 mg/ml (in the rangeof about 5.2 mg/ml and 6.1 mg/ml).

TABLE 5 Calculated concentrations of Thermoase ingredients in thesolubilization process; first step: Thermoase lyophilizate dissolved ina volume of 6.5 l, prior to addition of solid NaCl IngredientConcentration CaCl₂ 5 mM HEPES, pH 7.5 20 mM NaCl ~329 mM Na₂SO₄ ~5 mMSalts and buffer salts ~359 mM Crude thermolysin ~10 mg/ml Intactthermolysin ~7 mg/ml

TABLE 6 Calculated concentrations of salts including buffer salts in thesolubilization process; second step: 6.5 l volume, after addition ofsolid NaCl Ingredient Concentration CaCl₂ ~5 mM HEPES, pH 7.5 ~20 mMNaCl ~2790 mM Na₂SO₄ ~5 mM Salts and buffer salts ~2820 mM

TABLE 7 Calculated final concentrations of Thermoase and saltingredients in the solubilization process; third step: volume adjustedto 8 l Ingredient Concentration CaCl₂ 5 mM HEPES, pH 7.5 20 mM NaCl~2267 mM Na₂SO₄ ~4 mM Salts and buffer salts ~2296 mM Crude thermolysin~8.1 mg/ml Intact thermolysin ~5.7 mg/ml

Example 2 Stability of Solutions of Thermolysin with a Reduced NaClConcentration

Firstly, a solution of Thermoase lyophilisate was prepared using buffer(B) and the three-step procedure as described in Example 1. In asubsequent step, the solution was diafiltrated, wherein the buffer ofthe liquid composition was changed to 20 mM HEPES, 5 mM CaCl₂, 170 mMNaCl, pH 7.5. Surprisingly, thermolysin remained stabilized, i.e. thediafiltrated solution remained homogeneous for 5 hours and even longerthan 5 hours.

HPLC analysis of the solution of the Thermoase lyophilisate wasperformed before and after diafiltration using HPLC with a SUPERDEX 75pg 10 mm/300 mm GL column (GE Healthcare Bio-Sciences AB) as thestationary phase. The mobile phase was an aqueous buffer containing 200mM NaCl, 1 mM CaCl₂, 50 mM HEPES, pH 7.5. Sample volumes were 50 μleach. The flow rate was 0.5 ml/min, each HPLC run was performed for 80min. The detection unit was a UV-Vis absorbance detector operated at 280nm.

In each sample the main peak eluted with a mean retention time of about27 min 34 sec (see the peaks in area (i) shown in FIG. 2 and in area(vi) shown in FIG. 3).

Samples analyzed after the first step (no diafiltration) typicallyshowed peaks that could be grouped into five different areas as shown inFIG. 2. The area under the main peak (denoted (i)) reflectssubstantially undegraded (=intact) thermolysin. The peak shouldercorresponding to elution after a retention time of about 20 min 15 secwas assumed to reflect thermolysin dimers. The peaks under the areasdenoted (ii), (iii), and (iv) mainly corresponded to degradationproducts of thermolysin. Peaks under the area denoted (v) mainlyreflected more (to different degrees) strongly degraded thermolysinfragments as well as impurities.

Samples analyzed after the second step, i.e. following diafiltrationgave results as exemplarily shown in FIG. 3. Most notably, the relativeamount of degradation fragments and impurities corresponding to the peakareas denoted (vii) and (viii) were reduced in diafiltratedpreparations. In addition, the main peak in the area designated (vi)typically was much more distinct when compared with its counterpart inFIG. 2. Thus, the chromatogram after diafiltration indicated substantialseparation and purification of undegraded thermolysin.

TABLE 8 Characterization of peaks and quantification of the peak areasof FIG. 2 Retention Relative time Height Area peak area Peak area No.[min] [mA.U.] [mA.U. * min] [%] (i) 27.56 42.381 88.655 72.19 (ii) 31.330.679 2.658 2.16 (iii) 33.59 2.810 11.595 9.44 (iv) 38.60 3.074 7.6926.26 (v) 41.27 2.479 12.201 9.94 Σ 51.423 122.801 100.00

TABLE 9 Characterization of peaks and quantification of the peak areasof FIG. 3 Retention Relative time Height Area peak area Peak area No.[min] [mA.U.] [mA.U. * min] [%] (vi) 27.57 33.257 71.322 95.01 (vii)31.61 0.460 2.906 3.87 (viii) 50.39 0.418 0.837 1.11 Σ 34.135 75.065100.00

For further testing, two different stock solutions of thermolysin (i) 5mg/ml and (ii) 2.5 mg/ml were prepared, whereby at both concentrationsthe aqueous buffer was adjusted to 20 mM HEPES, 5 mM CaCl₂, 170 mM NaCl,pH 7.5 by way of diafiltration; the conductivity of the buffer with thedissolved thermolysin was about 20 mS/cm. Both solutions were sterilizedby sterile filtration and dispensed into aliquots.

From the time point when the lyophilisate was dissolved in the firstaqueous buffer until the aliquots of the two stock solutions wereobtained, thermolysin remained dissociated in solution for about 6 h.Both stock solutions remained clear throughout the process.

Additionally, an amount of 5 mg of Thermoase lyophilizate was dissolvedin 1 ml of an aqueous buffer containing 1 mM CaCl₂ and 5 mM HEPES, pH7.5. A 50 μl aliquot of the solution was analyzed by HPLC under standardconditions (see above) using a SUPERDEX 75 column 10/300, a pump speedof 0.5 ml/min and with UV/Vis detection at 280 nm. The mobile phase was1 mM CaCl₂, 200 mM NaCl, 50 in M HEPES, pH 7.5 in water. The thermolysinpeak was quantified relative to the other peaks obtained. Using thisapproach the relative amount of substantially intact thermolysin wasdetermined in several lots of Thermoase. Four independent determinationsindicated that on the average about 70% of the protein fraction ofThermoase was intact thermolysin. Individual values found were 59%, 72%,71%, and 76%.

An exemplary amount of 100 g of Thermoase lyophilisate contained about33 g of crude thermolysin, about 65 g of NaCl, and about 2 g of Na₂SO₄.Variation of protein content in Thermoase preparations was observed inthe range of 30 g and 35 g per 100 g of lyophilizate. Typically, thefraction of substantially undegraded and enzymatically activethermolysin with a molecular weight of about 34,600 Da (also referred toas “thermolysin”) was about 23% in the dry lyophilisate. The remainingamount of about 10% of protein in the dry lyophilisate mainly consistedof degradation products of thermolysin of which about 80% retainedproteolytic activity. The remainder comprised more heavily degradedthermolysin fragments and other impurities. Therefore, “crudethermolysin” in the sense of the invention is understood as being aprotein mixture consisting of (a) about 70% substantially undegradedthermolysin, (b) about 24% of thermolysin degradation products whichretain proteolytic activity (to different degrees), and (c) about 6% ofproteolytically inactive fragments and further impurities.

Example 3 Stability of Thermolysin Stock Solutions with a Reduced NaClConcentration at Different Temperatures

Aliquots of the two stock solutions of thermolysin with theconcentrations of (i) 5 mg/ml and (ii) 2.5 mg/ml obtained according tothe procedure of Example 2 (i.e. including diafiltration) were incubatedat different temperatures. In intervals of 30 min turbidity was assessedby visual inspection and measurements as described in Example 9. Thetime of exposure to the respective temperature before a solution becameturbid was recorded. The results are summarized in Table 10.

So far, all of the working steps were performed at 8° C. or lower, i.e.at temperatures in the range of 2° C. and 8° C. Both stock solutionsremained clear throughout the process.

TABLE 10 Thermolysin concentrations, temperatures and time during whichthe thermolysin solution remained clear Concentration of thermolysin in20 mM HEPES, 5 mM CaCl₂, 170 mM NaCl, pH 7.5 2.5 mg/ml 5 mg/ml Time ofpermanency of Temp. homogeneous (clear) solution 0° C. 21 h 8 h 2° C. 21h 8 h 4° C. 21 h 8 h 6° C. 21 h 8 h 8° C. 21 h 8 h 10° C.  21 h 4 h

Example 4 Stability of Solutions with a Thermolysin Concentration of 2.5Mg/Ml after Freezing and Thawing

After an incubation for 21 h at 4, 6, 8, and 10° C., aliquots containing2.5 mg/ml (see Example 3) were frozen at −20° C. and stored at thattemperature for 6 days. After thawing the aliquots were incubated at 8°C. The time of exposure to the temperature before a solution becameturbid was recorded. The results are summarized in Table 11.

TABLE 11 Incubation temperatures before freezing and time after thawingduring which the thermolysin solution remained clear Time of permanencyof homogeneous (clear) solution after Temp. before freezing thawing 4°C. 7 h 6° C. 6 h 8° C. 4 h 10° C.  1 h

Example 5 Stability of Solutions with Different Concentrations ofThermolysin after Freezing and Thawing

Stock solutions with thermolysin solutions in the range of 1 mg/ml and 5mg/ml were prepared similarly as described in Example 2. Aliquots of thestock solutions were frozen at −20° C. and stored at that temperaturefor 7 days. After thawing the aliquots were incubated at 8° C. The timeof exposure to the temperature before a solution became turbid wasrecorded. The results are summarized in Table 12.

TABLE 12 Thermolysin concentrations and time after thawing during whichthe thermolysin solution remained clear Concentration of thermolysin in20 mM HEPES, 5 mM CaCl₂, 170 mM NaCl, pH 7.5  1 mg/ml  2 mg/ml 2.5 mg/ml3 mg/ml 4 mg/ml   5 mg/ml Time of permanency of homogeneous (clear)solution 27 h 27 h 27 h  27 h 7 h 3.5 h

Example 6 Stability of Solutions Containing Thermolysin in the Presenceof Different Salts

Thermoase lyophilisate was dissolved at concentrations of 100, 50, and25 mg/ml in 20 mM HEPES, 5 mM CaCl₂ pH 7.5. Directly afterwards, a saltselected from the group consisting of Na₂SO₄, NaCH₃COO, NaCl, NaBr,NaNO₃, and NaJ was added and dissolved. As a control, no salt was added.Table 13 indicates the concentrations of the respective ions in thesolutions, taking into account the amounts present in the lyophilisate.Note that the ions present in the HEPES buffer are not accounted for inthe table.

TABLE 13 Solutions of thermolysin with different salts added CH₃CO O⁻,Cl⁻, conc. crude Br⁻, salt lyophilisate thermolysin thermolysin Na⁺ Cl⁻SO₄ ²⁻ NO₃ ⁻, J⁻ No. added in [mg/ml] in [mg/ml] in [mg/ml] ~[M] ~[M]~[M] [M] 1 — 100 ~33 ~23 1.125 1.122 0.006 0 2 50 ~16.5 ~11.5 0.5620.566 0.003 0 3 25 ~8.3 ~5.8 0.281 0.288 0.002 0 4 Na₂SO₄ 100 ~33 ~235.125 1.122 2.006 0 5 50 ~16.5 ~11.5 4.562 0.566 2.003 0 6 25 ~8.3 ~5.84.281 0.288 2.002 0 7 NaCH₃COO 100 ~33 ~23 3.125 1.122 0.006 2.0 8 50~16.5 ~11.5 2.562 0.566 0.003 2.0 9 25 ~8.3 ~5.8 2.281 0.288 0.002 2.010 NaCl 100 ~33 ~23 3.125 3.122 0.006 2.0 11 50 ~16.5 ~11.5 2.562 2.5660.003 2.0 12 25 ~8.3 ~5.8 2.281 2.288 0.002 2.0 13 NaBr 100 ~33 ~233.125 1.122 0.006 2.0 14 50 ~16.5 ~11.5 2.562 0.566 0.003 2.0 15 25 ~8.3~5.8 2.281 0.288 0.002 2.0 16 NaNO₃ 100 ~33 ~23 3.125 1.122 0.006 2.0 1750 ~16.5 ~11.5 2.562 0.566 0.003 2.0 18 25 ~8.3 ~5.8 2.281 0.288 0.0022.0 19 NaJ 100 ~33 ~23 3.125 1.122 0.006 2.0 20 50 ~16.5 ~11.5 2.5620.566 0.003 2.0 21 25 ~8.3 ~5.8 2.281 0.288 0.002 2.0

Table 14 indicates, with respect of the mixtures given in Table 13, ifand for how long a stable homogeneous (i.e. clear) solution wasobtained. Turbidity was assessed by visual inspection and measurementsas described in Example 9. The symbols presented in the table indicateas follows:

a.u., 90° side scatter assessment by visual inspection measurement {# ##} opaque 901->1000 (overflow) {0 0} cloudy 401-900 {o} slightly cloudy131-400 { } clear  0-130

TABLE 14 Stability of solutions of thermolysin in the presence ofdifferent salts No. salt added [0.1 h] [1 h] [5 h] [10 h] [15 h] [20 h]1 — {###} {###} {###} {###} {###}{###} >1000 >1000 >1000 >1000 >1000 >1000 2 {###} {###} {###} {###}{###} {###} >1000 >1000 >1000 >1000 >1000 >1000 3 {###} {###} {###}{###} {###} {###} >1000 >1000 >1000 >1000 >1000 >1000 4 Na₂SO₄ {###}{###} {###} {###} {###} {###} >1000 >1000 >1000 >1000 >1000 >1000 5{###} {###} {###} {###} {###} {###} >1000 >1000 >1000 >1000 >1000 >10006 {###} {###} {###} {###} {###}{###} >1000 >1000 >1000 >1000 >1000 >1000 7 NaCH₃COO {###} {###} {###}{###} {###} {###} >1000 >1000 >1000 >1000 >1000 >1000 8 {o} {o} {00}{###} {###} {###} 150 329 680 958 >1000 >1000 9 { } { } { } {o} {00}{00} 82 85 113 268 479 520 10 NaCl {###} {###} {###} {###} {###} {###}912 >1000 >1000 >1000 >1000 >1000 11 { } { } { } {o} {o} {00}/ 110 120126 189 302 507 12 { } { } { } { } { } { } 85 87 81 92 102 83 13 NaBr {} { } { } { } { } { } 110 116 114 95 104 112 14 { } { } { } { } { } { }95 110 125 85 93 85 15 { } { } { } { } { } { } 88 100 79 87 106 102 16NaNO₃ { } { } { } { } { } { } 75 85 103 102 88 86 17 { } { } { } { } { }{ } 68 65 73 69 100 79 18 { } { } { } { } { } { } 88 59 100 67 95 86 19NaJ { } { } { } { } { } { } 78 93 112 85 96 79 20 { } { } { } { } { } {} 112 105 126 102 98 115 21 { } { } { } { } { } { } 109 96 75 112 87 83Results are tabulated as {visual assessment} and “numerical value”(a.u., 90° side scatter measurement)

Example 7 Stability of Solutions Containing Thermolysin in the Presenceof Different Salts

Thermoase lyophilisate was dissolved at concentrations of 100, 50, and25 mg/ml in 20 mM HEPES, 5 mM CaCl₂ pH 7.5. Directly afterwards, a saltselected from the group consisting of KCl, NaCl, LiCl, MgCl₂, and CaCl₂was added and dissolved. As a control, no salt was added. Table 15indicates the concentrations of the respective ions in the solutions,taking into account the amounts present in the lyophilisate. Note thatthe ions present in the HEPES buffer are not accounted for in the table.

TABLE 15 Solutions of thermolysin with different salts added K⁺, Li^(+,)conc. crude Mg⁺⁺, salt lyophilisate thermolysin thermolysin Na⁺ Cl⁻ SO₄² ⁻ Ca⁺⁺ added in [mg/ml] in [mg/ml] in [mg/ml] ~[M] ~[M] ~[M] [M] 22 —100 33 23 1.125 1.122 0.006 0 23 50 16.5 11.5 0.562 0.566 0.003 0 24 258.3 5.8 0.281 0.288 0.002 0 25 KCl 100 33 23 1.125 3.122 0.006 2.0 26 5016.5 11.5 0.562 2.566 0.003 2.0 27 25 8.3 5.8 0.281 2.288 0.002 2.0 28NaCl 100 33 23 3.125 3.122 0.006 2.0 29 50 16.5 11.5 2.562 2.566 0.0032.0 30 25 8.3 5.8 2.281 2.288 0.002 2.0 31 LiCl 100 33 23 1.125 3.1220.006 2.0 32 50 16.5 11.5 0.562 2.566 0.003 2.0 33 25 8.3 5.8 0.2812.288 0.002 2.0 34 MgCl₂ 100 33 23 1.125 5.122 0.006 2.0 35 50 16.5 11.50.562 4.566 0.003 2.0 36 25 8.3 5.8 0.281 4.288 0.002 2.0 37 CaCl₂ 10033 23 1.125 5.122 0.006 2.0 38 50 16.5 11.5 0.562 4.566 0.003 2.0 39 258.3 5.8 0.281 4.288 0.002 2.0

Table 16 indicates, with respect of the mixtures given in Table 15, ifand for how long a stable homogeneous (i.e. clear, non-cloudy) solutionof thermolysin was obtained. Turbidity was assessed by visual inspectionand measurements as described in Example 9. The symbols presented in thetable indicate as follows:

a.u., 90° side scatter assessment by visual inspection measurement {# ##} opaque 901->1000 (overflow) {0 0} cloudy 401-900 {o} slightly cloudy131-400 { } clear  0-130

TABLE 16 Stability of solutions of thermolysin after different saltsadded salt added [0.1 h] [1 h] [5 h] [10 h] [15 h] [20 h] 22 — {###}{###} {###} {###} {###} {###} >1000 >1000 >1000 >1000 >1000 >1000 23{###} {###} {###} {###} {###} {###} >1000 >1000 >1000 >1000 >1000 >100024 {###} {###} {###} {###} {###}{###} >1000 >1000 >1000 >1000 >1000 >1000 25 KCl {###} {###} {###} {###}{###} {###} >1000 >1000 >1000 >1000 >1000 >1000 26 {o} {o} {00} {###}{###} {###} 250 394 599 >1000 >1000 >1000 27 { } { } { } {0} {00} {###}82 95 119 269 708 988 28 NaCl {###} {###} {###} {###} {###}{###} >1000 >1000 >1000 >1000 >1000 >1000 29 { } { } { } {o} {o} {00} 85115 129 189 350 487 30 { } { } { } { } { } { } 98 102 115 78 95 105 31LiCl { } { } { } { } { } { } 78 85 76 115 104 126 32 { } { } { } { } { }{ } 69 78 103 78 75 86 33 { } { } { } { } { } { } 88 96 84 109 83 87 34MgCl₂ { } { } { } { } { } { } 102 85 78 96 109 76 35 { } { } { } { } { }{ } 96 86 87 79 115 78 36 { } { } { } { } { } { } 84 76 95 88 94 117 37CaCl₂ { } { } { } { } { } { } 102 115 123 100 99 114 38 { } { } { } { }{ } { } 85 78 95 86 112 79 39 { } { } { } { } { } { } 78 79 72 85 110 95Results are tabulated as {visual assessment}/“numerical value” (a.u.,90° side scatter measurement)

Example 8 Determination of Turbidity

For the present invention, instrument-based observations of turbiditywere made with a CARY ECLIPSE instrument (Varian, Inc. Palo Alto,Calif., USA).

Two liquid samples were provided, the first being a homogeneous aqueousbuffer with 5 mM CaCl₂, 170 mM NaCl, 20 mM HEPES, pH 7.5 as dissolvedingredients; the second sample was a colloid consisting of the samebuffer and additionally about 5 mg/ml thermolysin (also see Examples 2and 3). Before measurements were taken, the colloid was allowed to formovernight at 10° C.

The parameter settings of the instrument were the following:

Instrument: Cary Eclipse Instrument serial number EL06033429 Data Mode:Fluorescence Scan Mode Emission X mode Wavelength (nm) Start (nm) 200Stop (nm) 1000 Ex. Wavelength (nm) 800 Ex. Slit (nm) 5 Em. Slit (nm) 5Scan rate (nm/min) 600 Data interval (nm) 1 Averaging Time (s) 0.1Excitation filter Auto Emission filter open PMT voltage (V) mediumCorrected spectra Off Multicell holder Multicell Multi zero Off

Liquid samples were analyzed in standard quartz cuvettes. Both sampleswere characterized by way of a wavelength scan, whereby the wavelengthof the incoming light was increased from 200 nm to 1,000 nm. Thedetection wavelength was kept constant at 800 nm. Neither fluorescencenor opalescence was detected. Scattered light was detected at thewavelength of the incoming light. Results are depicted in FIGS. 1 (A)and (B). For the clear buffer sample the measured intensity of scatteredlight was about 100 arbitrary units (a.u.). For the colloid of thesecond sample the scattered light created an overflow at the detector.

Example 9 Visual Inspection and Turbidity Measurements

For the demonstration of the effects of the present invention,instrument-based observations of turbidity were made with a CARY ECLIPSEinstrument (Varian, inc. Palo Alto, Calif., USA). The wavelength of theincoming light was 800 nm; 90° side scatter was measured at the samewavelength (i.e. 800 nm). The parameter settings of the instrument werethe following:

Instrument: Cary Eclipse Instrument serial number EL06033429 Data Mode:Fluorescence Em. Wavelength (nm) 800 Ex. Wavelength (nm) 800 Ex. Slit(nm) 5 Em. Slit (nm) 5 Ave Time (sec) 0.1 Excitation filter AutoEmission filter open PMT voltage (V) medium Multicell holder MulticellMulti zero Off Replicates 1 Sample averaging off

Liquid samples were analyzed in standard quartz cuvettes.

Visual inspection of thermolysin containing liquid samples in test tubeswas performed by grouping the samples into four categories: (i) “clear”,(ii) “slightly cloudy”, (iii) cloudy, (iv) “opaque”. Thus, categories(ii) to (iv) reflected increasing degrees of turbidity. The categorieswere correlated with the readout of arbitrary units (a.u.) as indicatedin Table 17.

TABLE 17 Turbidity of liquid samples, categories Category Exemplarymeasured values Value range for category clear 114; 85; 95  0-130slightly cloudy 182; 188; 236; 248; 304; 297 131-400 cloudy 760; 783;810; 815; 847 401-900 opaque ~1000; >1000 901->1000 (=overflow)

For the purpose of the present invention, a “clear” thermolysincontaining solution is characterized by a turbidity (determined asabove) which is about equivalent to (i.e. about equals) the turbidity ofa solution without thermolysin but otherwise with the same compositionand concentrations of the respective ingredients. In line with theinvention and as shown above, this corresponds to the range of 0 a.u.and 130 a.u., more preferred to the range of 50 a.u. and 130 a.u.,determined as 90° side scatter using light with a wavelength of 800 nmunder the conditions described above.

Example 10 Spectrophotometric Detection of Protein from ThermoasePreparation

All procedures prior to photometry were performed at ice-coldtemperature. Thermoase was provided as freeze-dried amorphous powder(Daiwa Kasei K.K.). An amount of Thermoase lyophilizate was dissolved inaqueous Tris buffer containing 10 mM CaCl2, 40 mM TrisHCl, pH 7.5 toyield a solution of Thermoase lyophilizate with a concentration of 1mg/ml.

As soon as a clear solution was obtained (assessed by visualinspection), photometric readings were taken at 277 nm and at 25° C.

Three different lots of Thermoase were analyzed repeatedly. Photometricreadings ranged from 0.57 to 0.61. The potential impact of differencesbetween A (1 mg/ml) values determined at 277 nm and 280 nm were assumedto be insignificant and smaller than other potential sources of error.

TABLE 18 Percentage [w/w] of protein (crude thermolysin) in Thermoasepreparations using different A (1 mg/ml) values as reference extinctionat 277 nm A (1 mg/ml) Thermoase Lot (range) 1.765 1.83 1 0.60-0.6134%-35% 33% 2 0.57-0.59 32%-33% 31%-32% 3 0.58-0.60 33%-34% 32%-33%

1. A method for preparing a solution of stabilized thermolysin (EC3.4.24.27), the method comprising: (a) the first step of mixing a solidpreparation comprising thermolysin with an aqueous solvent and making afirst solution, wherein the first solution comprises a first buffer saltcapable of maintaining a pH in the range of 4.5 to 9, one or more secondsalts, and thermolysin, wherein in the first solution the aggregateconcentration of the first and second salts is in the range of about 0.1mM to 500 mM, (b) the second step of adding to the first solution ameasured amount of a third salt, wherein the third salt is selected fromthe group consisting of NaCl, NaBr, NaNO₃, Nal, KCl, LiCl, MgCl₂, CaCl₂,and a mixture thereof, and (c) dissolving the third salt in the firstsolution, thereby obtaining a second solution in which the dissolvedthermolysin is in a stabilized form.
 2. The method according to claim 1,wherein in step (a) the solid preparation comprising thermolysinadditionally comprises one or more fourth salts.
 3. The method accordingto claim 1, wherein in step (a) the solid preparation comprisesthermolysin at a concentration in the range of about 20% to 100% (w/w).4. The method according to claim 1, wherein in step (a) theconcentration of the preparation comprising thermolysin in the aqueoussolvent is in the range of about 1 mg/ml to about 100 mg/ml.
 5. Themethod according to claim 1, wherein the first solution comprisessulfate ions and wherein the concentration of the sulfate ions in thefirst solution obtained in step (a) is in the range of about 1 mM toabout 10 mM.
 6. The method according to claim 5, wherein the totalconcentration of the third salt in the second solution obtained afterstep (b) is in the range of about 2 M to about 3.5 M.
 7. The methodaccording to claim 1, wherein in the second solution obtained after step(b) the concentration of thermolysin is between about 5 mg/ml and thanabout 35 mg/ml.
 8. The method according to claim 7, wherein in thesecond solution obtained after step (b) the concentration of thermolysinis between about 10 mg/ml and than about 35 mg/ml, and the third salt instep (b) is selected from the group consisting of NaCl, NaBr, NaNO₃,Nal, KCl, LiCl, MgCl₂, and CaCl₂.